MicroRNA-582-3p targeting ribonucleotide reductase regulatory subunit M2 inhibits the tumorigenesis of hepatocellular carcinoma by regulating the Wnt/β-catenin signaling pathway

ABSTRACT Hepatocellular carcinoma (HCC) is an important cause of death worldwide. MicroRNA (miRNA)-mediated gene silencing is involved in tumor biology. In this study, we aimed to elucidate the function and mechanism of action of miR-582-3p in HCC. We performed reverse transcription-quantitative polymerase chain reaction and western blotting to detect the expression levels of miR-582-3p, ribonucleotide reductase regulatory subunit M2 (RRM2), and markers of the Wnt/β-catenin signaling pathway (Wnt, Gsk-3β, β-catenin, and C-myc). The potential binding between miR-582-3p and RRM2 was confirmed using a dual-luciferase reporter assay. The proliferative and migratory capacities of the cells were evaluated using the cell counting kit-8 and wound-healing assays, respectively. Mouse models were used to validate the role of miR-582-3p in vivo. We observed the downregulation of miR-582-3p levels in HCC tumors and cell lines. Its upregulation in Huh7 and Hep 3B cells impaired their proliferation and migration, and the in vivo results showed suppressed tumor growth. Additionally, miR-582-3p upregulation also reduced the expression levels of Wnt, β-catenin, and C-myc, but enhanced the expression levels of glycogen synthase kinase-3β, both in vitro and in vivo. miR-582-3p targeted RRM2, and a negative correlation was observed in its expression patterns in HCC. Furthermore, RRM2 overexpression aggravated the proliferative and migratory capabilities of Hep3B and Huh7 cells and triggered Wnt/β-catenin signaling. However, miR-582-3p depleted RRM2 expression, thereby attenuating the oncogenic effects of RRM2. In conclusion, our results demonstrated that miR-582-3p binds to RRM2 to regulate the Wnt/β-catenin signaling pathway, thereby blocking the progression of HCC.


Introduction
Hepatocellular carcinoma (HCC) accounts for 75-85% of primary liver cancers [1]. Several risk factors, such as hepatitis B or C virus infection, aflatoxin exposure, smoking, and heavy alcohol intake contribute to HCC development [2]. HCC has a high mortality rate owing to its late diagnosis, frequent recurrence, and drug resistance. A five-year survival rate of > 50% has been observed among patients after surgical resection [3]. Screening and surveillance at an early stage for patients with HCC may improve therapeutic outcomes and five-year survivals [4]. Accordingly, emerging research has proposed promising biomarkers that contribute to early diagnosis and therapeutic monitoring of HCC, such as circulating tumor DNA and non-coding RNA [5,6]. Developing more potential biomarkers is essential to enhance the survival of patients' survival.
MicroRNAs (miRNAs) are short non-coding RNAs closely associated with various human diseases. The deregulation of miRNAs is linked to disease progression in different cancer types, including HCC [7]. Moreover, miRNAs are considered to serve as either oncogenic miRNAs or tumor suppressor miRNAs that govern multiple cellular processes during tumor growth [8]. It is well known that miRNAs modulate various molecular biological phenotypes by depleting the expression of miRNA target genes. miRNA dysregulation affects the activity of several molecular signaling pathways linked to carcinogenesis, such as mitogen-activated protein kinase (MAPK), winglessrelated integration site (Wnt), and Janus kinase/signal transducer and activator of transcription (JAK/STAT) [9]. In fact, a growing body of evidence suggests that abnormal Wnt/β-catenin signaling promotes HCC development and/or progression [10][11][12]. In the Wnt/β-catenin signaling pathway, when Wnt ligands are absent, signaling is inactive, and β-catenin is phosphorylated by a destruction complex that includes adenomatous polyposis coli protein (APC), AXIN, casein kinase I (CK1), and glycogen synthase kinase-3 (GSK-3β). Phosphorylated β-catenin is targeted for proteasomal degradation. When Wnt/β-catenin signaling is activated in the presence of Wnt ligands, a receptor complex forms between Frizzled and lipoprotein receptor-related protein families (LPR). Subsequent LRP6 phosphorylation leads to AXIN, GSK-3β, and Disheveled (DSH) recruitment, which blocks AXIN-mediated phosphorylation of β-catenin, thus preventing the formation of the destruction complex. In the nucleus, β-catenin promotes the expression of target genes by interacting with transcription factors and other proteins [11]. Thus, studying the association between various small molecules, miRNAs, and cancer progression is helpful for the development of cancer therapy and the discovery of cancer pathogenesis [13,14]. Among these miRNAs, miR-582-3p is involved in diverse cancers and exerts different functional effects [15,16]. A recent study revealed a marked downregulation of miR-582-3p in HCC tumors and cells, clearly suggesting its participation in HCC [17]. However, the precise mechanism underlying the function of miR-582-3p in HCC remains unclear.
As shown in the Gene Expression Profiling Interactive Analysis database, RRM2 is frequently overexpressed in multiple tumor tissues, including liver cancer. A previous study has defined RRM2 as a therapeutic target and prognostic biomarker for HCC. It was also observed in that study that restoring RRM2 expression recovered HCC cell growth [18]. However, the exact role of RRM2 in HCC remains unclear. Interestingly, using the bioinformatics tool starBase [19], miR-582-3p was predicted to contain binding sites with the RRM2 3′-untranslated region (UTR). This implied that RRM2 is miR-582-3p's putative target. However, the relationship between RRM2 and miR-582-3p has not been elucidated in previous studies.
Based on the above results, we speculated that the interplay between miR-582-3p and RRM2 might regulate HCC progression. Therefore, we determined the expression of miR-582-3p and RRM2 in HCC and explored the role and molecular mechanism of miR-582-3p in HCC using in vitro and in vivo experiments. The findings of this study may provide novel biomarkers for HCC management. In addition, we examined the effect of the miR-582-3p/RRM2 axis on the Wnt/βcatenin pathway in HCC.

Clinical samples
The patients were diagnosed with HCC at Wuhan Asia General Hospital. Prior to tissue excision, the patients enrolled in this study did not receive any antitumor therapy. All the participants provided written informed consent. During surgery, the tumors and adjacent normal tissues were excised and placed in liquid nitrogen. All tissue samples were preserved at −80°C. A total of 40 pairs of tumors and matched normal tissues were used for the analysis. This study was approved by the Ethics Committee of Wuhan Asia General Hospital (Approval number: 2021011).

Cell culture
The liver epithelial cell line (non-cancer controls), THLE2, and several HCC cell lines (Huh7, Hep 3B, SNU-182, and Hep10) were sourced from the BeNa Culture Collection (China). Following the protocol, THLE2, Hep 3B, Hep10, and Huh7 cell lines were inoculated into Dulbecco's modified Eagle's medium (DMEM; Sigma, USA) containing 10% fetal bovine serum (FBS; Sigma). On the other hand, the SNU-182 cell lines were propagated in a Roswell Park Memorial Institute (RPMI) 1640 Medium (Sigma) supplemented with 10% FBS. Finally, cell cultures were maintained at 37°C in a constant-temperature incubator filled with 5% CO 2 .

Cell transfection
Ribobio (China) was used to match mimic-NC and mimicked miR-582-3p. The RRM2 overexpression fusion vector (RRM2-OE) and pcDNA empty vector were provided by Sangon Biotech (Shanghai,China). Hep3B and Huh7 cell lines were plated and cultivated overnight in culture plates with 24 wells. Next, the vectors (1 μg) and mimics (40 nM) were transfected into these cells using the Lipofectamine 3000 Transfection Reagent (Invitrogen, USA). Western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were performed 24 h after transfection to examine transfection efficiency.

RT-qPCR
The cell or tissue samples were lysed using TRIzol reagent to obtain RNA. Subsequently, RNA was quantified using a NanoDrop2000 (Thermo Fisher Scientific, USA). Following the manufacturer's protocol, RNA samples (1 μg) were assembled into cDNA using a cDNA Synthesis Kit (NEB, USA) or an miRNA First-Strand cDNA Synthesis Kit (GeneCopeia, USA). Next, cDNA was diluted and amplified by qPCR using a Universal qPCR Master Mix (SYBR) (NEB). The reaction was performed on a StepOne™ Real-Time PCR System (Applied Biosystems, USA). GAPDH and U6 were used to normalize the data. Finally, the Ct value was calculated using the 2 −ΔΔCt approach [20]. Table 1 lists the primer information.

CCK-8 assay
The assay was adapted from a previous report [21]. Briefly, treated cells (5,000 cells/well) were inoculated into 96 wells culture plate in triplicate. The cells were maintained at 37°C for the indicated times. After 0, 24, 48, and 72 h, CCK-8 reagent (Sigma) was added to the cells to cultivate cells for two more hours. Finally, the absorbance of each well was measured at a wavelength of 450 nm using a microplate reader (Thermo Fisher Scientific). A cell viability curve was generated by plotting the optical density (OD 450 ) values against time.

Wound-healing assay
The assay was adapted from a previous report [21]. In triplicates, 50,000 treated cells per well were seeded into 24 wells culture plates. A sterile tip (200 μL) was used to scrape the cell surface and create an artificial wound. The cell phenotypes were photographed using a light microscope (Nikon, Japan). After culturing the cells for 24 h, the wound healing distance was observed again using a light microscope.
Finally, an enhanced chemiluminescence (ECL) kit (Beyotime) was used to visualize the protein signals.
All antibodies used in this study were acquired from Abcam (Cambridge,USA).

Xenograft tumor experiment
All procedures in this animal study were approved by Wuhan Asia General Hospital. Ten nude mice (6-8-week-old; female) sourced from Charles River (Beijing, China) were randomly assigned to two groups (n = 5). Hep3B cells were subcutaneously administered into nude mice to induce tumor formation. During tumor growth, either miR-582-3p agomiR (Ribobio) or NC agomiR (Ribobio) was administered to nude mice via intratumoral injections once a week. During this period, tumor length and width were gauged using a Vernier caliper to compute the tumor volume (length×width 2 × 0.5). Euthanasia was performed on all mice after 28 days, and the tumors were excised for additional analysis.
With the help of a Lipofectamine 3000 Transfection Reagent, Huh7 and Hep3B cells were co-transfected with either one of the RRM2 reporter vectors plus a miR-582-3p mimic or a mimic-NC. The cells were maintained for 48 h before they were examined for luciferase activity using a dual-luciferase reporter assay system (Promega, USA).

Statistical analysis
All experiments were independently conducted three times. GraphPad Prism 7 software (GraphPad Inc., USA) was used to process the data and generate figures. Student's t-test and analysis of variance were applied to determine whether the differences between the two groups and among multiple groups were significant. The association between the two variables was examined using the Pearson's test. P < 0.05 was deemed to be statistically significant.

Results
Recent studies have shown that miRNAs can be either oncogenic or tumor suppressors and govern multiple cellular processes during tumor growth. In this study, we aimed to elucidate the function and mechanism of action of miR-582-3p in HCC. We first assessed the levels of miR-582-3p in HCC tumors and cell lines using qRT-PCR. We performed rescue experiments by upregulating miR-582-3p to detect its effect on the malignant properties of HCC using functional assays (CCK-8 and wound healing assays). Additionally, we observed the expression of markers of the Wnt/β-catenin signaling pathway using western blotting to study the effect of miR-582-3p upregulation on Wnt/β-catenin signaling, both in vitro and in vivo. Furthermore, the association between miR-582-3p and RRM2 was predicted using bioinformatics and validated by dual-luciferase reporter gene assays, functional assays, and western blotting.

Upregulation of miR-582-3p restrains HCC cell proliferation and migration and blocks the Wnt/ β-catenin signaling pathway
We examined the miR-582-3p expression patterns and observed a noticeable downregulation in HCC tumors (Figure 1(a)). A pronounced decline in miR-582-3p expression was also observed in Huh7, SNU-182, Hep10, and Hep3B cells compared to that in THLE2 cells (Figure 1(b)). Since Huh7 and Hep3B cells had the lowest miR-582-3p expression, they were used in the functional assays. The expression level of miR-582-3p was considerably enhanced in Hep3B and Huh7 cells transfected with miR-582-3p mimic (Figure 1(c)). Functionally, the CCK-8 assay revealed that Hep3B and Huh7 cells overexpressing miR-582-3p exhibited notably impaired proliferative capacity (Figure 1(d)). Moreover, when compared to the NC, an inhibitory phenotype of cell migration was monitored among miR-582-3p-mimic-transfected Hep3B and Huh7 cells (Figure 1(e)). Additionally, the expression of proteins in the Wnt/β-catenin signaling pathway was quantified. The data showed notably reduced levels of Wnt, β-catenin, and c-myc in Hep3B and Huh7 cells with miR-582-3p upregulation. In contrast, p-Gsk-3β levels were enhanced (Figure 1(f)). These results implied that miR-582-3p inhibits the proliferation and growth of HCC cells, possibly by inactivating the Wnt/β-catenin signaling pathway.

miR-582-3p upregulation represses HCC tumorigenesis and Wnt/β-catenin signaling activity in the animal models
The role of miR-582-3p was further examined in animal models to determine whether its in vitro effects could translate into tumorigenesis in vivo. Injecting nude mice with Hep3B cells overexpressing miR-582-3p markedly decreased the weight and volume of the tumors (Figure 2(a-c)). Tumor imaging revealed poor tumor size in miR-582-3p-overexpressionadministered group. Furthermore, we observed reduced levels of RRM2, Wnt, β-catenin, and c-Myc in the tumors of nude mice injected with cells overexpressing miR-582-3p (Figure 2(d)). Conversely, the level of p-Gsk-3β was enhanced in these tumors. These results demonstrated that miR-582-3p impedes tumor growth, reduces RRM2 expression, and inactivates the Wnt/-catenin signaling pathway in vivo.

miR-582-3p targets RRM2 and shows negative correlation with RRM2 expression in HCC
Since miR-582-3p reduced RRM2 expression, we further explored its association with RRM2. StarBase analysis found that RRM2 3'-UTR had binding sites for miR-582-3p, thus implying that RRM2 was a prospective miR-582-3p target (Figure 3(a)). A MUT sequence fragment of the RRM2 3′-UTR was incorporated for the dual-luciferase reporter experiment. The results indicated that the combined transfection of miR-582-3p and RRM2 3′-UTR WT strikingly sequestered luciferase activity compared to RRM2 3′-UTR MUT co-transfection (Figure 3(b)). These results confirmed the binding between miR-582-3p and RRM2. The levels of RRM2 were substantially upregulated in HCC cell lines and tumors compared to those in normal THLE2 cell lines and tissues, respectively (Figure 3(c-d)). The expression of miR-582-3p was negatively associated with RRM2 expression in HCC tumor samples (Figure 3(e)). These observations clearly suggested that miR-582-3p targets RRM2.

miR-582-3p targets RRM2 to inhibit the effects of RRM2 on HCC cell growth and Wnt/β-catenin signaling activity
RRM2-OE mice were used to monitor the functional effects of RRM2. As a result, RRM2-OE markedly enhanced RRM2 expression compared to the empty vector. Moreover, the miR-582-3p mimic notably diminished RRM2 expression compared with mimic-NC. Co-transfection with OE +mimic partially recovered RRM2 expression compared to transfection with the mimic alone (Figure 4(a)). In terms of function, RRM2 overexpression prominently enhanced the proliferative capacities of Huh7 and Hep3B cells, whereas miR-582-3p upregulation showed the opposite effects. Meanwhile, introduction of the miR-582-3p mimic in RRM2-overexpressed cells largely repressed cell proliferation (Figure 4(b)). Moreover, migration was stimulated in Huh7 and Hep3B cells overexpressing RRM2. However, introducing the miR-582-3p mimic into these cells significantly weakened their migratory capacity (Figure 4(c)). Overexpression of RRM2 reinforced the expression levels of Wnt, β-catenin, and c-myc but reduced the expression level of p-Gsk-3β. In contrast, introducing the miR-582-3p mimic attenuated the effects induced by RRM2 (f) Relative protein expression levels of Wnt, β-catenin, C-myc, GSK-3β, and p-GSK-3β in miR-582-3p mimic-transfected cells was quantified via western blot experiment. B: *P < 0.05 and **P < 0.001 vs. THLE2; C-F: *P < 0.05 and **P < 0.001 vs. mimic-NC.

Discussion
The summarization of miRNA signatures has greatly promoted the development of tumor biology, and numerous miRNAs in HCC have been functionally illustrated [22][23][24]. Our research considered miR-582-3p as a study objective and reported that its upregulation restrained the proliferation and migration of HCC cells, while also hindering tumor development in vivo. RRM2, a newly identified miR-582-3p target, prompted a series of oncogenic effects in HCC. miR-582-3p impairs HCC malignant phenotypes by repressing RRM2, which is a novel mechanism that could contribute to our understanding of HCC pathogenesis.
Previous research deciphering the role of miR-582-3p in the progression of various cancers has been a subject of conflict. It exhibits poor expression in prostate cancer, prevents metastasis to bones, and blocks cancer cell migration and invasion [16]. Similarly, in hematologic malignancies, such as acute myeloid leukemia (AML), the expression levels of miR-582-3p are considerably reduced in the blood of patients with AML. In addition, miR-582-3p enrichment arrested the cell cycle of AML cells and retarded their proliferation [15]. Conversely, upregulation of miR-582-3p has been observed in hypoxia-related lung cancer, which promotes cancer cell proliferation and metastasis [25]. In the case of HCC, miR-582-3p has been demonstrated to be downregulated, and its overexpression reduces cell proliferation and induces cell cycle arrest in the G0/G1 phase by targeting CDK1 and AKT3, suggesting it to be a tumor suppressor [26]. Several reports have suggested that non-coding RNAs can function as miRNA sponges by competitively interacting with and inhibiting their downstream targets in HCC. Some studies have identified miR-582-3p as a potential miRNA target. Circular RNAs, such as circRNA_104075, circRNA_PTPRA, and circ_HIPK3, are overexpressed in HCC and stimulate HCC tumorigenesis by absorbing miR-582-3p [17,27]. Our findings were consistent with those of previous studies, and we observed downregulation of miR-582-3p in HCC cell lines and tumors. We found that miR-582-3p upregulation suppressed the proliferative and migratory capabilities of HCC cells in vitro and slowed the development of tumors in vivo. These findings highlight the role of miR-582-3p as a tumor suppressor in HCC.
The activation of Wnt/β-catenin signaling is widely monitored in human cancers, and its targeted inhibition has been proposed as a promising cancer therapy [28]. The aberrant activation of Wnt/β-catenin signaling induces aggressive proliferation, metastasis, and energy metabolism in cancer cells to accelerate tumorigenesis [29]. For example, miR-192 and miR-215 depletion decelerated gastric cancer cell proliferation and migration by reducing the activity of Wnt/β-catenin signaling [30]. Additionally, miR-504 enrichment hinders HCC cell growth and invasion by repressing Wnt/β-catenin signaling [31]. Here, we first determined the association between Wnt/β-catenin signaling and miR-582-3p expression. The data clearly showed that enriching miR-582-3p increased p-Gsk-3β levels while depleting Wnt, β-catenin, and C-myc levels. Notably, the serine threonine kinase Gsk-3β negatively modulates Wnt/βcatenin signaling by impairing β-catenin cytosolic stabilization; its activity is reflected by its phosphorylated form [32]. In this study, we demonstrated that miR-582-3p enhances p-Gsk-3β expression to impede the activation of Wnt/β-catenin signaling. miR-582-3p has numerous targets that have not yet been fully identified. We identified RRM2 as a novel miR-582-3p target. RRM2 is a well-studied oncogene that promotes cancer cell growth, migration, invasion, and chemoresistance in malignancies, such as retinoblastoma and breast, pancreatic, and renal cancers [33][34][35]. Carcinogenic effects of RRM2 have also been observed in HCC [18,36]. Consistent with previous reports, we observed that RRM2 overexpression aggravated HCC cell proliferation and migration. Interestingly, RRM2 is involved in miR-520a-mediated inactivation of the Wnt/β-catenin pathway during the development of non-small cell lung cancer [37]. RRM2 silencing has been suggested to inactivate the Wnt/β-catenin signaling pathway by increasing GSK-3β phosphorylation in multiple myeloma [38]. Our hypothesis that there is an interplay between miR-582-3p and RRM2 was confirmed by our findings, which demonstrated that miR-582-3p suppresses RRM2 expression to attenuate its carcinogenic effects. Moreover, RRM2 overexpression triggered Wnt/βcatenin pathway activation. Nevertheless, restoring miR-582-3p expression significantly weakened the activity of Wnt/β-catenin signaling.
Conclusion miR-582-3p is poorly expressed in HCC. Its upregulation inhibited HCC cell malignant behavior and in vivo tumorigenesis by impairing Wnt/β-catenin signaling via the sequestration of RRM2. However, the interactions between RRM2 and Wnt/β-catenin signaling need to be investigated further to determine the underlying role of RRM2 in HCC development, and to verify whether RRM2 regulates Wnt expression in HCC cells. Despite these limitations, our study offers new insights into miRNAbased therapies for HCC. In this study, we investigated the mechanism and role of miR-582-3p in HCC; however, its clinical implication requires further investigation. Moreover, further trials and studies are necessary to validate the clinical performance of miR-582-3p.

Availability of data and material
The datasets that have been used and/or analyzed during the study are available from the corresponding author upon reasonable request.